This procedure describes a method for isolation of murine hepatocytes using a modified two-step perfusion method. This protocol allows to measure the level of protein synthesis in primary hepatocytes after stimulation with growth factor and hormones. The advantage of this procedure is to easily perform a 35S-metabolic labelling to measure the level of protein synthesis upon stimulation ex vivo.

Authors: Valentina Gandin, Pier Carlo Marchisio and Stefano Biffo
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IL-17-expressing CD4+ cells (TH17 cells) constitute a considerable proportion of lymphocytes in the intestinal lamina propria (LP), even in healthy mice kept under specific pathogen free conditions. TH17 cells are rarely observed in the spleen, mesenteric lymph node, or Peyer’s patches. Germ-free mice or antibiotics (vancomycin and metronidazole)-treated mice show marked reductions in numbers of...

Authors: Koji Atarashi and Kenya Honda
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The lamina propria (LP) CD11c+ antigen-presenting cells directly sample the luminal contents and direct CD4+ T cells to differentiate into TH1, TH2 or TH17 cells (refs 1,2). Mouse CD11c+ LP cells consist of more than three subsets distinguished by different expression of cell surface markers, such as CD11b, CD70, CX3CR1, and CD103; and it has been reported that each subset has a distinct mission (...

Authors: Koji Atarashi and Kenya Honda
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Examination of utilized phosphorylation sites in an expressed, tagged protein requires a robust method that allows for maximum sequence coverage and identification of even low stoichiometry sites. The following protocol uses two to four enzymes to achieve high sequence coverage. Each of these digestions is analyzed un-enriched (C18) and enriched by TiO chromatography. By using this strategy and t...

Authors: Nicholas Sherman and Jay Fox
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We have developed a high-throughput protein purification protocol that allows us to purify ~4,000 proteins within 10 hours from prepared culture. By combining steps of cell lysis and protein capture on affinity resins in sealed filter plates, we reduced the number of pipetting steps and thus, human errors.

Authors: Chien-Sheng Chen and Heng Zhu
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Apoptotic cells, via the activation of caspases, undergo significant proteome alterations. Phagocytosis of apoptotic cells by dendritic cells (DCs) leads to the processing of the apoptotic cell-associated (apoptotic) antigens and the cross-presentation of the resulting peptides on major histocompatibility complex class I molecules. This phenomenon seems crucial for inducing either cross-priming or...

Authors: Pisana Moroni Rawson et al
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In vitro Treg suppression assays are performed to determine the functional effect of Treg cells on CD4 T cells. They are performed by co-culturing the responding population (Tresp) with the Treg cells or control CD4 cells (Tcon cells).

Authors: Pushpa Pandiyan
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Here we describe the efficient and facile synthesis of N-acetyl glucosamine propargyl O-glycoside, a compound which has been prepared previously by conventional methods[1]. In previous syntheses the glycoside was obtained as a mixture of anomers1a) and in moderate to acceptable yield1b). The described improved method exploits a high yielding and strongly β-selective lanthanide triflate catalysed[...

Authors: Henrik H Jensen, Holger B Kramer and Benjamin G Davis
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C-glycosides have received considerable attention in synthetic carbohydrate chemistry due to their unique features. As opposed to their natural congeners they are not prone to enzymatic or chemical hydrolysis. They can be prepared as anomerically pure glycosides and are isosteric to the heteroatom containing counterparts.[1] Here we describe the synthesis of an alkynyl C-galactoside which was succ...

Authors: Holger Kramer and Benjamin G Davis
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Methanethiosulfonates (MTS) are a group of reagents which allow the site-selective modification of accessible cysteine residues in proteins.[1] Synthesis of compounds carrying the MTS moiety has allowed the covalent attachment of structures to protein surfaces which represent functional mimics of post-translational modifications.[2] Here we describe the synthesis of ethyl-linked O-glycosyl methan...

Authors: Holger Kramer and Benjamin G Davis
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